A highly selective and sensitive turn-on chemodosimeter for hypochlorous acid based on an iridium(iii) complex and its application to bioimaging

Article abstract of DOI:10.1039/c4dt00616j

A novel and easily accessible luminescent iridium(iii) complex [Ir(tpy)₂(N^N)]PF₆ (Ir-ANMM, tpy = 2-(p-tolyl)pyridine, N^N = 4-[(4-amino-3-nitrophenoxy)-methylene]-4′-methyl-2,2′- bipyridine) for the sensing of HOCl has been designed and synthesized. The detection strategy is based on the HOCl-promoted cleavage of the PET quenching 4-amino-3-nitrophenyloxy moiety of the weakly emissive complex Ir-ANMM, which gives rise to a highly luminescent complex [Ir(tpy)₂(N^N′)] PF₆ (Ir-HM, N^N′ = 4-hydroxymethyl-4′-methyl-2,2′- bipyridine). Comparisons of the absorption, emission and mass spectra of Ir-ANMM in the absence and presence of HOCl have all confirmed the transformation of Ir-ANMM to Ir-HM. Titration and competition experiments have revealed high sensitivity and high selectivity of Ir-ANMM for HOCl, respectively. Significantly, the feasibility of the sensor under physiological conditions enables the successful luminescent imaging of HOCl in HeLa cells.

Full text of DOI:10.1039/c4dt00616j

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A highly selective and sensitive turn-on
chemodosimeter for hypochlorous acid based on
an iridium(III) complex and its application to
bioimaging†
Cite this: Dalton Trans., 2014, 43,
9529
Fengniu Lua and Tatsuya Nabeshima*a,b
A novel and easily accessible luminescent iridium(III) complex [Ir(tpy)2(N^N)]PF6 (Ir-ANMM, tpy =
2-(p-tolyl)pyridine, N^N = 4-[(4-amino-3-nitrophenoxy)-methylene]-4’-methyl-2,2’-bipyridine) for the
sensing of HOCl has been designed and synthesized. The detection strategy is based on the HOCl-pro-
moted cleavage of the PET quenching 4-amino-3-nitrophenyloxy moiety of the weakly emissive complex
Ir-ANMM, which gives rise to a highly luminescent complex [Ir(tpy)2(N^N’)]PF6 (Ir-HM, N^N’ = 4-hydroxy-
methyl-4’-methyl-2,2’-bipyridine). Comparisons of the absorption, emission and mass spectra of
Ir-ANMM in the absence and presence of HOCl have all confirmed the transformation of Ir-ANMM to
Ir-HM. Titration and competition experiments have revealed high sensitivity and high selectivity of
Ir-ANMM for HOCl, respectively. Significantly, the feasibility of the sensor under physiological conditions
enables the successful luminescent imaging of HOCl in HeLa cells.
Received 28th February 2014,
Accepted 10th April 2014
DOI: 10.1039/c4dt00616j
www.rsc.org/dalton
quently,
a number of iridium(III) complexes have been
Introduction
employed as luminophores for sensing various analytes.7
Despite the significance of HOCl sensing and the attractive
Hypochlorous acid (HOCl) is a reactive oxygen species (ROS)
and is widely employed in our daily lives as a bactericide and a photophysical characteristics of iridium(III) complexes, there
bleaching agent.1 As a normal byproduct of cellular metabo- has been only one example of a hypochlorite sensor based on
lism,2 endogenous HOCl plays essential roles in many vital an iridium complex reported thus far.8 The sensor, taking
biological processes. However, concentrated hypochlorite solu- advantage of the oxidation of oxime, was capable of detecting
tions can pose a great threat to health because excessive HOCl hypochlorite. However, it suffers from poor water solubility
may result in tissue damage and numerous diseases.3 The con- due to the instability of oxime in aqueous media, which has
tribution of HOCl to both health and disease has strongly pro- undoubtedly limited its practical application. As a result, there
moted the monitoring of HOCl and elucidation of its remains a high demand for the development of practically
functional mechanisms both in vivo and in vitro. Many fluo- applicable HOCl sensors based on the favorable iridium(III)
rescent sensors for HOCl have been reported in recent years.4 complexes.
However, such organic fluorophore-based sensors always
Recently, the reaction between the o-nitroanilino group and
suffer from a small Stokes shift and severe photobleaching.5 HOCl9 has been successfully employed for the selective detec-
Conversely, luminescent iridium(III) complexes exhibit advan- tion of HOCl.10 Based on this reaction, we designed and syn-
tageous photophysical properties such as good photostability, thesized an easily accessible chemodosimeter Ir-ANMM
a large Stokes shift, high emission efficiency and sensitive substituted with an o-nitroanilino group for HOCl sensing
optical properties to the chemical environment.6 Conse- (Scheme 1). The sensor Ir-ANMM should possess very weak or
no emission with the PET effect imposed by the electron-rich
o-nitroanilino group. However, in the presence of HOCl, the
o-nitroanilino group could be oxidized, followed by the cleavage
aGraduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba,
Ibaraki 305-8571, Japan. E-mail: nabesima@chem.tsukuba.ac.jp;
Fax: +81-29-853-4507; Tel: +81-29-853-4507
of the 4-amino-3-nitrophenyloxy moiety and generation of a
hydroxymethyl group appended complex Ir-HM, which,
without the PET effect, should exhibit strong luminescence.
Such a turn-on emission response can be well utilized for the
sensing of HOCl. With an amino group and a hydroxyl group,
respectively, both Ir-ANMM and Ir-HM should possess appro-
bTsukuba Research Center for Interdisciplinary Materials Science (TIMS), University
of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4dt00616j
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Scheme 1 The design concept of Ir-ANMM.
priate water solubility, enabling the detection of HOCl in was recorded using an MM-60R instrument (TDADKK). The
aqueous media, which is a prerequisite of the sensor for practi- UV-vis spectra were recorded on a JASCO V-660 spectro-
cal applications. Moreover, a reaction-based chemodosimeter photometer. The fluorescence spectra and absolute quantum
such as Ir-ANMM should possess very high selectivity.11
yields were measured on a Hitachi F-4500 spectrometer and a
As anticipated, in the presence of HOCl, the weak emission Hamamatsu Photonics absolute PL quantum yield measure-
of Ir-ANMM was remarkably enhanced. The absorption, emis- ment system C9920-02, respectively. The ESI-TOF mass spectra
sion and ESI mass spectral comparisons of Ir-ANMM in were recorded on an Applied Biosystems QStar Pulsar i spectro-
the absence and presence of HOCl have all confirmed the meter and a Waters SYNAPT G2 system. Elemental analyses
transformation of Ir-ANMM to Ir-HM. Significantly, the titra- were performed at the Chemical Analysis Center, University of
tion experiments indicated an extremely low detection limit, Tsukuba. All bright-field imaging and luminescence imaging
and the competition experiments demonstrated a highly measurements were carried out on an Olympus FV-1000 micro-
specific luminescent response to HOCl among various reactive scope. The luminophore in HeLa cells was excited with a high-
oxygen species (ROS) and reactive nitrogen species (RNS). pressure Hg lamp through a 460–490 nm filter, and emission
These properties, together with the applicability of the sensing was collected over 500 nm. The image processing was per-
under physiological conditions, make Ir-ANMM capable of formed using the QCaptuer Pro 6.0 software.
imaging HOCl in living cells, which is of striking importance
for intracellular HOCl sensing in view of the significant bio-
logical impact of HOCl.
Synthesis
ANMM-bpy: A mixture of 4-amino-3-nitrophenol (925 mg,
6.00 mmol) and NaH (144 mg, 6.00 mmol) was stirred in
60 mL of dry acetonitrile at room temperature under an argon
atmosphere. After 15 min, a solution of BM-bpy (526 mg,
2.00 mmol) in 20 mL of dry acetonitrile was injected. The sus-
pension was stirred overnight and then filtered to remove the
precipitate. The filtrate was evaporated and purified by flash
column chromatography (SiO2; CH2Cl2–acetone, 10 : 1, v/v) to
give ANMM-bpy as an orange powder. Yield: 74%, m.p.
Experimental section
Materials
All starting materials and reagents, unless otherwise specified,
were purchased from commercial suppliers and used without
further purification. 4-Bromomethyl-4′-methyl-2,2′-bipyridine
(BM-bpy),12 4-hydroxymethyl-4′-methyl-2,2′-bipyridine (HM-
1
202–205 °C. H NMR (400 MHz, CDCl3) δ (ppm): 8.69 (d, J =
bpy),13 and [Ir(tpy)2Cl]2 (tpy = 2-(p-tolyl)pyridine) were syn-
14
5.2 Hz, 1H), 8.55 (d, J = 5.2 Hz, 1H), 8.46 (s, 1H), 8.26 (s, 1H),
7.65 (d, J = 2.8 Hz, 1H), 7.38 (d, J = 4.8 Hz, 1H), 7.19 (d, J = 9.2
Hz, 1H), 7.16 (d, J = 4.8 Hz, 1H), 6.79 (d, J = 9.2 Hz, 1H), 5.92
(s, 2H), 5.13 (s, 2H), 2.45 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ (ppm): 156.75, 155.61, 149.49, 149.33, 149.05, 148.26, 146.54,
140.29, 131.48, 126.98, 124.94, 122.11, 121.54, 120.27, 119.12,
108.16, 69.21, 21.21; positive-ion ESI-MS m/z calcd for
[C18H17N4O3]+ ([M + H]+): 337.1, found: 337.0.
thesized and purified according to the reported procedures. All
reactions were performed under a nitrogen or an argon atmos-
phere (where noted). Column chromatography was performed
using Kanto Chemical silica gel 60 N (spherical, neutral). De-
ionized water was freshly prepared from a water purification
system (MILIPORE) and used throughout. The human female
cervix tumor cell line, HeLa (RCB0007), was provided by the
RIKEN BioResource Center.
Ir-HM: A suspension of [Ir(tpy)2Cl]2 (113 mg, 0.10 mmol)
and HM-bpy (40 mg, 0.20 mmol) in CH2Cl2 and CH3OH
(30 mL, 1 : 1, v/v) was heated to reflux while stirring under a
Apparatus
Melting points were determined on a Yanaco melting point nitrogen atmosphere. After 12 h the resulting yellow solution
apparatus and not corrected. The 1H NMR spectra were was cooled to room temperature. The solution volume was
recorded on a Bruker AV400 spectrometer at 400 MHz. The 13C then reduced to approximately 15 mL and ammonium hexa-
NMR spectra were recorded on a Bruker AV400 spectrometer at fluorophosphate (5-fold excess, aq., 10 mL) was added. After
100 MHz. For the 1H and 13C NMR measurements, tetra- stirring for 2 h, the mixture was evaporated and the residue
methylsilane (TMS) was used as the internal standard. The pH was dissolved in CH2Cl2 (30 mL). The solution was washed
9530 | Dalton Trans., 2014, 43, 9529–9536
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with water and brine and then dried over MgSO4. After fil- added, and the final volume was adjusted to 5 mL with phos-
tration, the solvent was removed under vacuum. The crude phate buffer. All samples were allowed to stand at room tem-
product was purified by column chromatography (SiO2; perature for at least 3 h before measurement. Luminescent
CH2Cl2–acetone, 20 : 1, v/v) and Ir-HM was obtained as a spectra were obtained in 10 × 10 mm quartz cells with the exci-
yellow powder. Yield, 94%, m.p. 270–273 °C. 1H NMR tation at 400 nm.
(400 MHz, CD3CN) δ (ppm): 8.46 (s, 1H), 8.39 (s, 1H), 7.98 (d,
J = 8.0 Hz, 2H), 7.88 (d, J = 5.6 Hz, 1H), 7.81–7.77 (m, 3H), 7.67
Luminescent imaging of HeLa cells
(d, J = 8.0 Hz, 2H), 7.54 (d, J = 6.0 Hz, 2H), 7.42 (d, J = 6.0 Hz,
1H), 7.31 (d, J = 5.6 Hz, 1H), 6.96 (d, J = 6.0 Hz, 2H), 6.86 (d, J =
8.0 Hz, 2H), 6.09 (s, 2H), 4.78 (d, J = 4.2 Hz, 2H), 3.72 (t, J = 5.6
Hz, 1H), 2.52 (s, 3H), 2.09 (s, 6H); 13C NMR (100 MHz, CD3CN)
δ (ppm): 168.56, 156.71, 156.42, 152.87, 151.90, 151.86, 151.00,
150.73, 149.80, 142.36, 141.42, 139.20, 133.22, 129.87, 126.24,
126.10, 125.74, 124.31, 123.77, 122.23, 120.35, 62.61, 21.69,
21.36; positive-ion ESI-MS m/z calcd for [C36H32IrN4O]+ ([M –
PF6]+): 729.2, found: 729.2; elemental analysis calcd (%) for
C36H32IrN4OPF6·H2O: C 48.48, H 3.84, N 6.28; found: C 48.67,
H 3.98, N 5.96.
HeLa cells were seeded on glass-bottomed dishes and cultured
in Dulbecco’s modified Eagle’s medium (DMEM) for 24 h at
37 °C in a 5% CO2–95% air incubator. The concentrated stock
solution of Ir-ANMM (1 mM) was prepared by dissolving
Ir-ANMM in DMSO. Before cell loading, the cultured HeLa cells
were washed twice and then incubated with Ir-ANMM (final
concentration: 20 μM) in fresh cell culture medium. After incu-
bation for 1 h at 37 °C in a 5% CO2–95% air incubator, the
cells were washed three times with fresh DMEM media and
further incubated with or without HOCl (final concentration:
50 μM) for 0.5 h. The cells were rinsed three times with fresh
DMEM before being subjected to luminescence microscopy
imaging measurement.
Ir-ANMM:
A
suspension of [Ir(tpy)2Cl]2 (113 mg,
0.10 mmol) and ANMM-bpy (67 mg, 0.20 mmol) in CH2Cl2
and CH3OH (30 mL, 1 : 1, v/v) was heated to reflux while stir-
ring under a nitrogen atmosphere. After 12 h the resulting
solution was cooled to room temperature. The solution volume
was then reduced to approximately 15 mL and ammonium
hexafluorophosphate (5-fold excess, aq., 10 mL) was added.
After stirring for 2 h, the mixture was evaporated and the
residue was dissolved in CH2Cl2 (30 mL). The solution was
washed with water and brine and then dried over MgSO4. After
filtration, the solvent was removed under vacuum. The crude
product was purified by column chromatography (SiO2;
CH2Cl2–acetone, 20 : 1, v/v) and Ir-ANMM was obtained as an
Theoretical calculations
All the calculations based on the density functional theory
(DFT) were carried out using the Gaussian 09 program
package.15 The hybrid Hartree Fock/density functional theory
(HF/DFT) method B3LYP was utilized to optimize the geometry
of the ground state. The “double-ζ” quality basis set consisting
of Hay and Wadt’s effective core potentials (LANL2DZ) was
employed for the treatment of the iridium(III) atom, while the
6-31G* basis set was used for the treatment of the H, C, O and
N atoms. A relativistic effective core potential (ECP) replaced
the inner core electrons of IrIII, leaving the outer core (5s25p6)
electrons and the 5d6 valence electrons. Based on these
optimizations, 40 ground-to-excited state singlet–singlet tran-
sition energies (20 for Ir-HM) and 3 excited state triplet tran-
sition energies were obtained to determine the vertical
excitation energies of Ir-ANMM and Ir-HM using the time-
dependent DFT (TD-DFT) calculations.
1
orange powder. Yield, 96%, m.p. >300 °C. H NMR (400 MHz,
CD3CN) δ (ppm): 8.56 (s, 1H), 8.42 (s, 1H), 7.98 (d, J = 7.6 Hz,
2H), 7.94 (d, J = 5.6 Hz, 1H), 7.82–7.77 (m, 3H), 7.67 (d, J = 7.6
Hz, 2H), 7.61 (d, J = 2.8 Hz, 1H), 7.54 (d, J = 5.6 Hz, 2H), 7.51
(dd, J = 5.6, 1.6 Hz, 1H), 7.33 (dd, J = 5.6, 1.6 Hz, 1H), 7.26 (dd,
J = 9.2, 2.8 Hz, 1H), 6.98–6.94 (m, 3H), 6.86 (dd, J = 8.0, 1.2 Hz,
2H), 6.39 (s, 2H), 6.09 (d, J = 4.8 Hz, 2H), 5.24 (s, 2H), 2.54 (s,
3H), 2.09 (s, 6H); 13C NMR (100 MHz, CD3CN) δ (ppm): 168.55,
157.17, 156.21, 152.96, 151.72, 151.39, 151.20, 150.81, 149.82,
149.20, 142.70, 142.35, 141.49, 139.26, 133.19, 131.55, 130.06,
127.85, 126.75, 126.43, 125.77, 124.39, 123.80, 122.91, 121.61,
120.40, 109.18, 69.23, 21.71, 21.40; positive-ion ESI-MS
Results and discussion
Synthesis and characterization
m/z calcd for [C42H36IrN6O3]+ ([M − PF6]+): 865.2, found: The synthetic procedures of Ir-ANMM and Ir-HM are shown in
865.3; elemental analysis calcd (%) for C42H36IrN6O3PF6· Scheme S1.† 4-Bromomethyl-4′-methyl-2,2′-bipyridine (BM-
CH2Cl2·(CH3)2CO: C 47.92, H 3.85, N 7.29; found: C 48.26, bpy),12 4-hydroxymethyl-4′-methyl-2,2′-bipyridine (HM-bpy)13
H 3.83, N 7.41.
and [Ir(pba)2Cl]2 14 were synthesized and purified according to
the reported procedures. The ligand 4-[(4-amino-3-nitrophe-
noxy)-methylene]-4′-methyl-2,2′-bipyridine (ANMM-bpy) was
General procedures for luminescent sensing experiments
Unless otherwise noted, all luminescent sensing measure- synthesized through the nucleophilic substitution of BM-bpy
ments were carried out at room temperature in CH3CN–0.1 M by 4-amino-3-nitrophenol in dry CH3CN in the presence of
1
phosphate buffer (1 : 4, v/v, pH 7.4). Specifically, to a 5 mL NaH. The ligand was characterized by H NMR, 13C NMR and
volumetric flask, 0.1 mL of the stock solution (2.5 × 10−4 M) of ESI-MS. The two complexes Ir-ANMM and Ir-HM were syn-
Ir-ANMM in CH3CN was added, followed by the addition of thesized through the bridge splitting reaction of [Ir(tpy)2Cl]2
0.9 mL CH3CN and 2 mL phosphate buffer (0.1 M, pH 7.4). and subsequent complexation with ANMM-bpy and HM-bpy,
Then an appropriate volume of the ROS/RNS (see ESI†) was respectively. The complexes were both purified through
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column chromatography, with Ir-ANMM isolated as an orange DFT calculations
powder in 96% yield, while Ir-HM was isolated as a yellow
The TD-DFT calculations were carried out to acquire the rele-
vant transition energies of Ir-ANMM and Ir-HM, aiming for a
complete apprehension of the photophysical properties of
both complexes. The optimized structures of Ir-ANMM and
Ir-HM in the ground states are shown in Fig. S2,† based on
which the absorption transitions of both complexes have been
calculated. Fig. S3† shows an excellent accordance of the calcu-
lated absorption transitions of Ir-ANMM and Ir-HM with their
experimental spectra, confirming the applicable description of
these complexes by this calculation level. Additionally, the cal-
culated results in Tables S2 and S4† agree well with the former
ascription of the intense absorption bands below 335 nm pre-
dominantly to LC (Ir-ANMM: S30, S38, S39; Ir-HM: S15, S17,
S20) transitions and the absorption bands above 335 nm to an
powder in a yield of 94%. Both Ir-ANMM and Ir-HM were
characterized by 1H NMR, 13C NMR, ESI-MS and elemental
analysis.
Photophysical properties
The UV-vis absorption and photoluminescent spectra of
Ir-ANMM and Ir-HM are shown in Fig. 1 and the photophysical
data are summarized in Table S1.† In the electronic absorption
spectra, both complexes exhibit intense absorption bands with
extinction coefficients on the order of 104 dm3 mol−1 cm−1 in
the wavelength region shorter than 335 nm. These bands, by
comparison with the absorption characteristics of related bis-
cyclometalated iridium(III) diimine complexes,16 can be
ascribed to the ligand-centered (LC) (1IL of π→π*(C^N) and
1LLCT of π(C^N)→π*(N^N)) transitions. The less intense
absorption bands in the 335–492 nm (335–444 nm for Ir-HM)
region with extinction coefficients on the order of 103 dm3
mol−1 cm−1 are assigned to a mixture of the spin-allowed
metal-to-ligand charge-transfer (1MLCT) (dπ(Ir)→π*(C^N and
N^N)) transitions and ligand-to-ligand charge-transfer (1LLCT)
(π(C^N)→π*(N^N)) transitions. Such an ascription is further
confirmed by the TD-DFT calculations (vide infra).
1
admixture of LLCT–1MLCT (Ir-ANMM: S2, S8, S9, S10; Ir-HM:
S2, S4, S7, S9) transitions.
According to Table 1, the lowest triplet (T1) states of
Ir-ANMM and Ir-HM both originate from the HOMO–LUMO
(Ir-ANMM: 98.0%; Ir-HM: 98.2%) transitions. As shown in
Table 2, Tables S3 and S5,† the calculated HOMO of both com-
plexes mainly resides on the iridium center (Ir-ANMM: 41.8%;
Ir-HM: 42.0%) and the LUMO of both are dominantly located
on the bipyridine ligand (Ir-ANMM: 91.1%; Ir-HM: 94.5%).
Accordingly, both HOMO–LUMO transitions of Ir-ANMM and
Ir-HM are predominantly assigned to the 3MLCT (dπ(Ir)→π*-
(bpy)) characteristics, which are consistent with the previous
attribution. With similar MLCT units though, Ir-ANMM exhi-
bits a much lower emission quantum yield than Ir-HM, which
should originate from the PET effect imposed by its electron-
rich 4-amino-3-nitrophenyl moiety.10
As recorded in Table S1,† in aerobic acetonitrile, Ir-ANMM
and Ir-HM both exhibited weak emission with low quantum
yields of 0.01 and 0.04, respectively. In contrast, under anaero-
max
bic conditions, weak emission of Ir-ANMM (λ
= 619 nm,
em
max
Φem = 0.02) and efficient yellow emission of Ir-HM (λ
=
em
611 nm, Φem = 0.17) were observed. The extremely weak emis-
sion of Ir-ANMM confirmed the efficient PET effect imposed
by the electron-rich 4-amino-3-nitrophenyl moiety. The highly
oxygen-sensitive quantum yields suggested that the emission
arises from a triplet state. Moreover, the emission bands of
both Ir-ANMM and Ir-HM are dependent on the solvent
polarity (Fig. S1†), suggesting the 3MLCT (dπ(Ir)→π*(bpy and
C^N)) dominated excited states of the complexes,17 which was
further verified by the TD-DFT calculation studies.
Optimization of sensing conditions
To explore the possibility of sensing HOCl in aqueous media,
the effect of water on the emission of Ir-ANMM and Ir-HM was
investigated. As shown in Fig. S6,† introduction of various
amounts of water exerted very small effects on the lumine-
scence of Ir-ANMM, with constantly weak emission in diverse
solvent systems. Similarly, small amounts of water also have
little influence on the emission of the complex Ir-HM, leading
to a slight enhancement of its emission intensity. However,
with a large ratio of water, over 90% in particular, the emission
intensity dramatically reduced, which is definitely a serious
disadvantage since the distinct emission intensity gap of
Ir-ANMM and Ir-HM is necessary for the sensitive detection of
HOCl in such an emission turn-on sensing system. Taking into
account the disadvantageous influence of a large amount of
water on the emission of Ir-HM and the significance of detect-
ing analytes in aqueous media, CH3CN–H2O (1 : 4, v/v) was
chosen as an appropriate solvent system for the sensing of
HOCl.
The appropriate water solubility makes the sensor promis-
ing for intracellular applications. Accordingly, the applicability
of the sensor to biological systems was explored by studying
the influence of pH on the emission of Ir-ANMM and Ir-HM.
Fig. 1 The UV-vis absorption (solid lines) and PL emission (dash lines)
spectra of Ir-ANMM (5 μM) (blue) and Ir-HM (5 μM) (red). CH3CN. λex
400 nm. Inset: emission photographs of Ir-ANMM and Ir-HM.
=
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Table 1 Calculated excited states of Ir-ANMM and Ir-HM
Complex
State
Transition
Contri.
E/nm (eV)
Assignment
Ir-ANMM
Ir-HM
T1
T1
HOMO → LUMO
HOMO → LUMO
98.0%
98.2%
612 (2.02)
593 (2.09)
3MLCT/3LLCT
3MLCT/3LLCT
Table 2 Calculated molecular orbitals of Ir-ANMM and Ir-HM
Ir-ANMM Ir-HM
LUMO
HOMO
Fig. 2 The UV-vis absorption (solid lines) and PL emission (dash lines)
spectra of Ir-HM (5 μM) (red), Ir-ANMM (5 μM) in the absence (blue) and
presence (black) of 5 equiv. of HOCl. CH3CN–0.1 M phosphate buffer
(1 : 4, v/v, pH 7.4). λex = 400 nm. Inset: photographs of the color (top)
and PL emission (bottom) of Ir-HM (20 μM) and Ir-ANMM (20 μM) before
and after the addition of 5 equiv. of HOCl.
As shown in Fig. S7,† pH has negligible influence on the emis-
sion of both complexes in a range from pH 4.5 to pH 10.5,
indicating that the sensor is suitable for use under physiologi-
cal conditions. Based on these results, pH 7.4 was selected for
all the sensing experiments.
Typically, for a chemodosimeter, a fast response is a very
important factor for practical applications. Therefore, the
effect of time on Ir-ANMM sensing of HOCl was investigated
under the above optimized conditions, i.e. in a CH3CN–0.1 M
phosphate buffer (1 : 4, v/v, pH 7.4) solvent system. As shown
in Fig. S8,† the emission intensity of Ir-ANMM was dramati-
cally enhanced immediately after the addition of 5 equiv. of
HOCl, suggesting the feasibility of HOCl sensing by Ir-ANMM
in the solvent system. Significantly, the reaction reached com-
pletion within 20 min as monitored by the emission intensity
changes, indicating a relatively fast response of Ir-ANMM
toward HOCl.
intensity was enhanced dramatically and the solution emitted
strong yellow light. The absorption and emission spectra, solu-
tion color and emission color of the Ir-ANMM–HOCl mixture
were all very similar to those of Ir-HM, strongly implying the
HOCl-promoted oxidation of Ir-ANMM to generate Ir-HM in
this solvent system.
1
In addition to the optical spectral changes, H NMR spec-
troscopy was also employed to provide direct evidence of the
assumed sensing mechanism. As shown in Fig. S9,† in the
presence of HOCl, the 1H NMR spectrum of Ir-ANMM changed
strikingly. The disappearance of H6″ and H4″ indicated the clea-
vage of the 4-amino-3-nitrophenyloxy moiety. The upfield
shifts of H3, H5, H6, Hj and the spectral similarity to that of
Ir-HM have unambiguously confirmed the production of
Ir-HM from the reaction between Ir-ANMM and HOCl.
Moreover, the mechanism was further confirmed by ESI-MS
spectral changes. As shown in Fig. S10a,† the chemodosimeter
Ir-ANMM showed a featured m/z peak of [Ir-ANMM – PF6]+ at
865.3. However, the peak disappeared in the presence of
4 equiv. of HOCl (Fig. S10b†), with the appearance of a new m/z
peak at 729.2 characteristic of [Ir-HM – PF6]+ (Fig. S10c†). All
To ensure thorough reaction, all samples were measured
after standing at room temperature for at least 3 hours for all
the sensing experiments.
Optical response of Ir-ANMM to HOCl
The photophysical responses of Ir-ANMM toward HOCl under these results strongly demonstrated the HOCl-promoted oxi-
the optimized conditions are shown in Fig. 2. With higher dation reaction of Ir-ANMM to generate Ir-HM.
absorbance in the visible range, the solution of Ir-ANMM was
Sensitive response of Ir-ANMM to HOCl
pale yellow. In the presence of HOCl, however, the absorbance
in the visible range decreased and the solution color became The detailed luminescent response of Ir-ANMM toward HOCl
colorless.
has been carefully investigated by phosphorescence emission
The luminescence exhibits a much more pronounced spectroscopies. As shown in Fig. 3a, in the absence of HOCl,
response. In the absence of HOCl, Ir-ANMM showed very weak Ir-ANMM exhibits very weak emission. However, upon treat-
emission. Nevertheless, in the presence of HOCl, the emission ment with an increasing concentration of HOCl, the weak
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Fig. 4 Emission intensity I620 of Ir-ANMM (5 μM) in the presence of
various ROS/RNS. HOCl (25 μM), ONOO− (50 μM), and other (200 μM).
CH3CN–0.1 M phosphate buffer (1 : 4, v/v, pH 7.4). λex = 400 nm.
I620, while ONOO− resulted in a 4.5-fold enhancement and all
the other species generated negligible emission enhancement
(<2.6-fold). These results clearly demonstrated the high speci-
ficity of Ir-ANMM toward HOCl. Such a superior selectivity for
HOCl was attributed to the strong oxidizing capability of
HOCl, which could promote the oxidation of the o-nitroanilino
group in Ir-ANMM.
Fig. 3 (a) The luminescent spectral changes of Ir-ANMM (5 μM) upon
increasing addition of HOCl (from 0.0 to 25.0 μM). CH3CN–0.1 M phos-
phate buffer (1 : 4, v/v, pH 7.4). λex = 400 nm. (b) The emission intensity
I620 vs. the concentration of HOCl; inset: the linear relation of I620 vs.
the concentration of HOCl in the range of 0.0–15.0 μM.
Intracellular imaging of HOCl
The feasibility of the sensor under physiological conditions,
the high selectivity and the high sensitivity strongly supported
the examination of the applicability of Ir-ANMM for the deter-
mination of HOCl in living cells. Therefore, the feasibility of
Ir-ANMM for imaging HOCl in HeLa cells was evaluated. As
shown in Fig. 5a, the Ir-ANMM-loaded cells exhibit negligible
intracellular luminescence. In sharp contrast, the Ir-ANMM-
loaded cells further incubated with HOCl resulted in a remark-
able enhancement of the red intracellular luminescence
(Fig. 5b). These results indicated that Ir-ANMM was cell mem-
brane permeable and capable of interacting with HOCl to
generate measurable luminescent signals in the living cells.
emission was gradually enhanced and reached saturation
when 4 equiv. of HOCl was added. The titration curve was
depicted in Fig. 3b in which the emission intensity I620
increased in proportion to the HOCl concentration. Interest-
ingly, the emission intensity I620 showed an excellent linear
relationship with the HOCl concentration in the range of 0 to
15 μM (inset of Fig. 3b), indicating the feasibility of Ir-ANMM
for the quantitative determination of the HOCl concentration.
The detection limit, calculated from the linear equation, is 16
nM at the signal to noise ratio (S/N) = 3, which is much lower
than many reported luminescent HOCl sensors18 and therefore
confirms the highly sensitive response of Ir-ANMM to HOCl.
Selective response of Ir-ANMM to HOCl among various
ROS/RNS
The specific response of Ir-ANMM to HOCl was verified
through examination of the emission spectroscopic changes of
Ir-ANMM upon the addition of various ROS/RNS including
•
t
−
H2O2, OH, 1O2, O2−•, BuOOH, ROO•, NO•, NO2−, NO3 and
ONOO−. As shown in Fig. S11,† Ir-ANMM exhibited a remark-
able emission enhancement only in the presence of HOCl.
Conversely, in the presence of other ROS/RNS, even with a
large excess, negligible or only slight variations of the emission
spectra could be observed. The selectivity toward HOCl was
assessed by the emission intensity I620 changes. As shown in
Fig. 5 Images of HOCl in HeLa cells. (a) Images of the cells incubated
with Ir-ANMM (20 μM) only. (b) Images of Ir-ANMM-loaded cells after
Fig. 4, HOCl led to a 38-fold increase of the emission intensity treatment with HOCl (50 μM).
9534 | Dalton Trans., 2014, 43, 9529–9536
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Conclusions
A novel and easily accessible phosphorescent biscyclometa-
lated iridium(III) complex Ir-ANMM exhibiting a turn-on lumi-
nescent response to HOCl in aqueous media has been
synthesized and characterized. Upon the addition of HOCl, the
electron-rich PET-quenching 4-amino-3-nitrophenyl moiety of
the non-luminescent Ir-ANMM was cleaved, giving rise to a
new highly luminescent complex Ir-HM. The remarkable emis-
sion enhancement is accordingly able to indicate HOCl.
Through luminescent titration experiments, the limit of detec-
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specific reaction makes the chemodosimeter Ir-ANMM highly
selective for HOCl among various ROS/RNS. With appropriate
water solubility, low detection limit and high selectivity, the
probe was successfully employed for living cell imaging of
HOCl, making it a very promising tool for intracellular HOCl
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Acknowledgements
This research was financially supported by Grants-in-Aid for 10 Y. Xiao, R. Zhang, Z. Ye, Z. Dai, H. An and J. Yuan, Anal.
Scientific Research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan.
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